The present invention relates to ion sources utilizing ion-ion and ion-droplet chemical reactions to modify the charge-state distributions of ions generated by field desorption methods and in particular relates to ion sources that provide adjustable control of ion charge-state distributions produced by electrospray ionization.
Over the last several decades mass spectrometry has advanced to the point where it has become one of the most broadly applicable analytical tools to provide fast, sensitive and selective detection of a wide variety of molecules and ions. While mass spectrometric detection provides an effective means for identifying a wide variety of molecules, its use for analyzing high molecular weight compounds is currently hindered by problems related to producing gas phase ions attributable to a given analyte species. In particular, the application of mass spectrometric analysis to determine the composition of mixtures of important biological compounds, such as oligonucleotides and oligopeptides, is severely limited by experimental difficulties related to low sample volatility and unavoidable fragmentation during vaporization and ionization processes. As a result of these limitations, the potential for quantitative analysis of samples containing biopolymers via mass spectrometry remains largely unrealized. For example, the analysis of complex mixtures of DNA molecules produced in enzymatic DNA sequencing reactions is dominated by time-consuming and labor-intensive electrophoresis techniques that may be compromised by secondary structures. The ability to selectively and sensitively detect components of complex mixtures of biological compounds via mass spectrometric methods would aid considerably in improving the accuracy, speed and reproducibility of DNA sequencing methodologies and eliminate interferences arising from secondary structure. It would also open new possibilities for the characterization of complex mixtures of proteins, carbohydrates and other polymeric species.
To be detectable via mass spectrometric methods, a compound of interest must first be produced in the form of a gas phase ion. Accordingly, it is the ion formation process which largely dictates the scope, applicability and limitations of mass spectrometry. Conventional ion preparation methods for mass spectrometric analysis have proven unsuitable for high molecular weight compounds. Vaporization by sublimation and/or thermal desorption is unfeasible for many high molecular weight compounds, including biopolymers, because these species tend to have negligibly low vapor pressures. Ionization methods based upon the desorption process, which consists of emission of ions from solid or liquid surfaces, have proven more effective in generating ions from thermally labile, nonvolatile compounds. While conventional ion desorption methods, such as plasma desorption, laser desorption, fast particle bombardment and thermospray ionization, are more applicable to nonvolatile compounds, these methods suffer from substantial problems associated with ion fragmentation and low ionization efficiencies for compounds with high molecular masses (molecular mass greater than 2000 Dalton). To expand the applicability of mass spectrometric methods to samples containing biological compounds current research efforts have been directed toward developing new desorption and ionization methods suitable for high molecular weight species. As a result of these research efforts, two ion preparation techniques have evolved for the analysis of large molecular weight compounds; matrix assisted laser desorption and ionization-mass spectrometry (MALDI-MS) and electrospray ionization-mass spectrometry (ESI-MS).
MALDI and ESI ion preparation methods have profoundly expanded the role of mass spectrometry for the analysis of nonvolatile high molecular weight compounds including many compounds of biological interest. These ionization techniques provide high ionization efficiencies (ionization efficiency=(ions formed)/(molecules consumed)) and have been demonstrated to be applicable to biomolecules with molecular weights exceeding 100,000 Daltons. In MALDI, analyte is integrated into a crystalline organic matrix and irradiated by a short (≈10 ns) pulse of UV laser radiation at a wavelength resonant with the absorption band of the matrix molecules. Analyte molecules are entrained into a resultant gas phase plume and ionized via gas-phase proton transfer reactions occurring within the plume. While MALDI generally produces ions in singly and/or doubly charged states, significant fragmentation of analyte molecules during vaporization and ionization considerably limits the utility of MALDI as a source of gas phase ions directly attributable to a given parent compound. In addition, the sensitivity of the technique is dramatically affected by sample preparation methodology and the surface and bulk characteristics of the site irradiated by the laser. As a result, MALDI analysis is primarily used to identify the molecular masses of components of a sample and yields little information pertaining to the concentrations or molecular structures of materials analyzed.
In contrast, ESI is a field desorption ionization method that generally provides a means of generating gas phase ions with little interference from analyte fragmentation [Fenn et al., Science, 246, 64-70 (1989)]. Further, ESI provides an output consisting of a highly reproducible, continuous and homogeneous stream of analyte ions and is easily coupled to on-line liquid phase separation techniques such as high performance liquid chromatography (HPLC) and capillary electrophoresis. It is currently believed that field desorption ionization occurs by a mechanism involving strong electric fields generated at the surface of a charged substrate which extract solute analyte ions from solution into the gas phase. In ESI, a solution containing a solvent and an analyte is pumped through a capillary orifice maintained at a high electrical potential and directed at an opposing plate held near ground. The field at the capillary tip charges the surface of the emerging liquid and results in a stream of charged droplets. Subsequent evaporation of the solvent promotes a sequence of Coulombic explosions that results in droplets with a radius of curvature small enough that the electric field at their surface is large enough to desorb analyte species existing as ions in solution. Polar analyte species may also undergo desorption and ionization during electrospray by associating with cations and anions in the solution. Similar to ESI techniques, other field desorption methods have evolved that can successfully prepare ions from non-volatile, thermally liable, high molecular weight compounds. These techniques differ primarily in the physical manner in which the charged droplets are produced and include aerospray ionization, thermospray ionization and the use of pneumatic nebulization devices.
Since the ionization process proceeds via the formation of highly charged liquid droplets, ions produced by conventional field desorption methods such as ESI invariably possess a variety of multiply charged states for every analyte species discharged. Accordingly, ESI-MS spectra of mixtures are typically a complex amalgamation of peaks attributable to a large number of populated charged states for every analyte present in the sample. Therefore, ESI-MS spectra often possess too many overlapping peaks to permit effective discrimination and identification of the various components of a complex mixture. As a result of this limitation, the use of ESI-MS to analyze mixtures of biopolymers is currently severely hampered.
Recently, research efforts have been directed at expanding the utility of ESI-MS techniques for the analysis of complex mixtures of biopolymers. One method of reducing the spectral complexity of ESI-MS spectra uses computer algorithms that transform experimentally derived multiply charged spectra to xe2x80x9czero chargexe2x80x9d spectra [Mann et al., Anal. Chem., 62, 1702 (1989)]. While transformation algorithms take advantage of the precision improvement afforded by multiple peaks attributable to the same analyte species, spectral complexity, detector noise and chemical noise often result in missed analyte peaks and the appearance of false, artifactual peaks. However, the utility of transformation algorithms for interpreting ESI-MS spectra of mixtures of biopolymers may be substantially improved by manipulating the charge-state distribution of analyte ions produced in ESI and/or by operating under experimental conditions providing high signal to noise ratios [Stephenson and McLucky, J. Mass Spectrom. 33, 664-672 (1998)].
Alternatively, the complexity of ESI-MS spectra of mixtures of biopolymers may be reduced by operating the electrospray in a manner that decreases the net number of charge-states populated for a particular analyte compound. The ability to controllably reduce charge-state distributions to the extent that predominantly singly and/or doubly charged ions are formed would result in an ESI ion source well suited for the mass spectrometric analysis of high molecular weight compounds including biopolymers. A variety of methods of charge reduction have been attempted with varying degrees of success.
Griffey et al. report that the charge-state distribution of analyte ions produced by ESI may be manipulated by adjusting the chemical composition of the solution discharged [Griffey et al., J. Am. Soc. Mass Spectrom., 8, 155-160 (1997)]. They demonstrated that modification of solution pH and/or the abundance of organic acids or bases in a solution may result in ESI-MS spectra of oligonucleotides primarily consisting of singly and doubly charged ions. In particular, Griffey et al. report a decrease in the average charge-state observed in the electrospray of solutions of a 14 mer DNA from xe2x88x927.2 to xe2x88x923.8 upon addition of ammonium acetate to achieve a concentration of approximately 33 mM. Although altering solution conditions may improve the ease in which ESI spectra are interpreted, it does not allow for controllable charge reduction of all species present in solution. In addition, manipulation of solution composition may compromise ionization and/or transmission efficiencies in the electrospray ionization process.
An alternative approach to control the charge-state distribution of ions produced by ESI is to utilize gas phase chemical reactions of reagent ions to reduce the ionic charges of droplets and/or gas phase analyte ions generated upon electrospray discharge. This approach has the advantage of decoupling ionization and charge reduction processes to provide independent control of charge-state distribution. While independent control of charge reduction provides flexibility in choosing the sample buffer composition and the ESI operating conditions, practical constraints have limited its applicability to the analysis of mixtures of biopolymers.
To achieve a reduction in the charge-state distribution generated in the electrospray discharge of a solution containing a mixture of proteins, Ogorzalek et al. merged the output of an electrospray discharge with a stream of reagent ions generated using an externally housed Corona discharge [Ogorzalek et al., J. Am. Soc. Mass Spectrom., 3, 695-705 (1992)]. In particular, Ogorzalek et al. observed a decrease in the most abundant cation observed in the electrospray discharge of solutions containing horse heart cytochrome c from a charge state of +15 to a charge state of +13 upon merging a stream of anions formed via corona discharge. While the authors report a measurable reduction in analyte ion charge state distribution, generation of a population consisting predominantly of singly and/or doubly charged ions was not achievable.
Pui et al. (U.S. Pat. No. 5,992,244) also report a method for neutralizing charged particles purported to minimize particle losses to surfaces. In this method, charged droplets and/or particles are generated via electrospray and exposed to a flowing stream of oppositely charged electrons and/or reagent ions flowing in a direction opposite to that of the electrospray discharge. The authors describe the use of a neutralization chamber with one or more corona discharges distributed along the housing for producing free electrons and/or ions for neutralizing the output of an electrospray discharge. Electrically biased, perforated metal screens or plates are positioned along the housing of the neutralization chamber between the corona discharges and a neutralization region to create a confined electric field to conduct reagent ions toward the electrospray discharge. In addition, Pui et al., describe a similar charged particle neutralization apparatus in which the corona discharge ion source is replaced with a radioactive source of ionizing radiation for generating reagent ions. In both methods, neutralization is reported to reduce wall losses and enhance neutral aerosol throughput to an optical detection region located downstream of the electrospray discharge.
Stephenson et al., report a method of charge reduction in which singly charged reagent ions are generated by an externally housed glow discharge ion source and injected into the resonance cavity of an ion trap mass spectrometer containing oppositely charged neutralizing analyte ions generated by electrospray discharge [Stephenson and Mc Lucky, J. Mass Spectrom., 33, 664-672 (1998)]. Subsequent gas phase ion-ion reactions between analyte ions and reagent ions within the resonance cavity of the ion trap spectrometer are utilized to reduce the charge-state distribution of analyte ions. While Stephenson et al. report substantial charge reduction, instrumental constraints considerably restricted the range of analyte ion mass to charge ratios (m/z) useable for a given reagent ion. This limitation arises from the need to simultaneously constrain analyte ions and reagent ions to the spacial region within the cavity of the ion trap spectrometer to provide efficient reduction of the charge-state distribution. Accordingly, ion trap charge reduction techniques are less suitable for analysis of mixtures comprising high molecular weight biopolymers with a broad range of molecular masses. In addition, ion trap charge reduction devices are relatively expensive and not easily adaptable to pre-existing commercial ESI systems.
Gas phase reactions between the charged droplet output of an electrospray discharge and bipolar ions generated by a radioactive source have also been reported to affect the charge-state distributions of analyte ions generated in ESI. Zarrin et al. (U.S. Pat. No. 5,076,097) utilized radioactive Polonium strips positioned downstream of an electrospray discharge to convert the highly charged output of the electrospray discharge into a stream of neutral particles prior to optical characterization. The authors report that alpha particles emitted by the radioactive strips result in the formation of a gas comprised of both positively and negatively charged ions capable of neutralizing the particle stream formed upon discharge. By minimizing the loss of charged particles on the walls of the apparatus, the authors suggest that the use of their technique results in greater neutral particle throughput to a optical detection region located downstream of the electrospray discharge.
Kaufman et al. (U.S. Pat. No. 5,247,842) report an apparatus for producing uniform submicrometer droplets that utilizes a method of charge neutralization employing one or more radioactive Polonium strips positioned proximate to an electrospray discharge. The authors teach positioning a radioactive strip proximate to the electrospray, such that the droplets encounter the ions virtually immediately upon their formation. This placement is purported to be crucial in order to avoid droplet disintegration under Coulombic forces by rapidly neutralizing the droplets virtually immediately upon formation. In addition, the authors report a method of charge reduction in which a radioactive Polonium strip is placed upstream of an electrospray discharge apparatus to provide a flowing source of bipolar ions to the electrospray chamber. Finally, Kaufman et al. also suggest that a similar charge neutralization may be possible by positioning other sources of biopolar ions, such as a corona discharge or a source of ultraviolet radiation, proximate to the outlet of an electrospray discharge.
Scalf et al. also report a method of charge reduction that utilizes gas phase reactions of ions formed by a radioactive Polonium disk located downstream of the electrospray discharge [Scalf et al., Anal. Chem, 72, 52-60 (2000)]. Multiply charged analyte ions formed by the electro spray discharge undergo ion-ion chemical reactions that result in a decrease in the charge state distribution. Upon charge reduction, the analyte ions are pulsed into the evacuated flight tube of a time of flight mass spectrometer and detected with a multichannel plate. The authors report that the charge-states of ions produced by electrospray discharge of a liquid sample containing a mixture of proteins may be adjusted to yield predominantly singly and/or doubly charged ions attributable to each species in the sample. While this technique successfully reduces the spectral complexity of ESI-MS spectra, the necessity of a radioactive ion source significantly inhibits the commercial application of the technique due to stringent regulations pertaining to the use of radioactive materials.
It will be appreciated from the foregoing that a need still exists for a method of regulating the charge-state distribution of ions generated in ESI and other field desorption techniques to permit the mass spectral analysis of mixtures containing high molecular weight compounds. The present invention provides exemplary use of a corona discharge ion source located downstream of an electrospray discharge or other field desorption ion source to provide charge reduction. In particular, the present invention provides adjustable control of analyte ion charge-state distributions applicable to either operating polarity of an electrospray ionization apparatus.
The present invention provides methods and devices for generating ions from liquid samples containing chemical species, including but not limited to chemical species with high molecular masses. The methods and devices of the present invention provide an output comprising a continuous or pulsed stream of gas phase analyte ions of either positive polarity, negative polarity or both possessing either a selected fixed charge-state distribution or one that may be selectively varied with time. More specifically, the present invention provides ion sources with adjustable control of the charge-state distribution of the gas phase analyte ions generated.
In one embodiment, an ion source of the present invention comprises a flow of bath gas that conducts the output of an electrically charged droplet source through a field desorption-charge reduction region cooperatively connected to the electrically charged droplet source and positioned at a selected distance downstream with respect to the flow of bath gas. In this embodiment, either positively or negatively charged gas phase analyte ions of a selected charge state distribution are generated from liquid samples containing analytes. First, the electrically charged droplet source generates a continuous or pulsed stream of electrically charged droplets by dispersing a liquid sample containing at least one chemical species in at least one solvent, carrier liquid or both into a flow of bath gas. The droplets formed may possess either positive or negative polarity corresponding to the desired polarity of ions to be generated. Next, the stream of charged droplets and bath gas is conducted through a field desorption-charge reduction region where solvent and/or carrier liquid is removed from the droplets by at least partial evaporation to produce a flowing stream of smaller charged droplets and multiply charged gas phase analyte ions. Evaporation of positively charged droplets results in formation of gas phase analyte ions with multiple positive charges and evaporation of negatively charged droplets results in formation of gas phase analyte ions with multiple negative charges. The charged droplets, analyte ions or both remain in the field desorption-charge reduction region for a selected residence time controllable by selectively adjusting the flow rate of bath gas and/or the length of the field desorption region.
Within the field desorption-charge reduction region, the stream of smaller charged droplets and/or gas phase analyte ions is exposed to electrons and/or gas phase reagent ions of opposite polarity generated from bath gas molecules by a reagent ion source positioned at a selected distance downstream of the electrically charged droplet source. The reagent ion source is surrounded by a shield element for substantially confining the boundaries of electric fields and/or electromagnetic fields generated by the reagent ion source. In a preferred embodiment, the shield element is grounded. In an alternate preferred embodiment, the shield element is electrically biased and held at an electric potential close to ground. In a more preferred embodiment the shield element is held at approximately 250 V or approximately xe2x88x92250 V.
The shield element defines a shielded region wherein electric and/or electromagnetic fields are minimized. In a preferred embodiment the field desorption-charge reduction region is within the shielded region. Minimizing the extent of electric fields and/or electromagnetic fields in the field desorption-charge reductive region is desirable to minimize deflection of gas phase analyte ions, charged droplets or both by electric and/or electromagnetic fields. Accordingly, minimizing the presence of electric and/or electromagnetic fields is beneficial for maximizing the analyte ion throughput of the field desorption-charge regulation region.
Electrons, reagent ions or both, generated by the reagent ion source, react with charged droplets, analyte ions or both within at least a portion of the field desorption-charge reduction region and reduce the charge-state distribution of the analyte ions in the flow of bath gas. Accordingly, ion-ion, ion-droplet, electron-ion and/or electron-droplet reactions result in the formation of gas phase analyte ions having a selected charge-state distribution. In a preferred embodiment, the charge state distribution of gas phase analyte ions is selectively adjustable by varying the interaction time between gas phase analyte ions and/or charged droplets and the gas phase reagent ions and/or electrons. In addition, the charge-state of gas phase analyte ions may be controlled by adjusting the rate of production of electrons, reagent ions or both from the reagent ion source. In addition, an ion source of the present invention is capable of generating an output consisting of analyte ions with a charge-state distribution that may be selected or may be varied as a function of time.
In an exemplary embodiment, an ion source of the present invention comprises a field desorption-charge reduction region substantially free of electric fields and/or electromagnetic fields generated by the reagent ion source. Minimizing the extent of electric and/or electromagnetic fields in the field desorption-charge reduction region is beneficial because it prevents unwanted loss of charged droplets and/or ions on the walls of the apparatus and allows for efficient collection of ions generated by the ion source of the present invention. However, as the droplets and analyte ions are themselves electrically charged, maintaining a field desorption-charge reduction region completely free of electric fields is not possible.
The generation of electrically charged droplets in the present invention maybe performed by any means capable of generating a continuous or pulsed stream of charged droplets from liquid samples containing chemical species. In an exemplary embodiment, an electrospray ionization source is employed in which sample is pumped through an orifice held at a high electric potential and directed at an opposing metal plate held near ground. The potential difference between the orifice and metal plate is sufficiently high to create an electric field at the surface of the emerging liquid to disperse it into a fine spray consisting of charged droplets. Applying a positive electric potential to the orifice results in formation of positively charged droplets while selection of a negative electric potential results in formation of negatively charged droplets. Other electrically charged droplet sources useful in the present invention include, but are not limited to: nebulizers, pneumatic nebulizers, thermospray vaporizers, cylindrical capacitor generators, atomizers, and piezo-electric pneumatic nebulizers.
The generation of electrons and/or reagent ions in the present invention maybe performed by any means capable of generating electrons and/or reagent ions from bath gas molecules. In an exemplary embodiment, the reagent ion source generates electric fields and/or electromagnetic fields. In a preferred exemplary embodiment, the reagent ion source comprises a corona discharge positioned at a selected distance downstream of the electrically charged droplet source. In a more preferred embodiment, the corona discharge is selectively positionable at any point downstream of the electrically charged droplet source. The corona discharge comprises a first electrically biased element and a second element held at ground or substantially close to ground. The first and second elements are positioned sufficiently close to create a self-sustained electrical gas discharge. In this embodiment, the first electrically biased element may be held at either a positive voltage or a negative voltage. First and second corona discharged elements may have an adjustable potential difference ranging from approximately 10,000 V to approximately xe2x88x9210,000 to provide control of the abundance of gas phase reagent ions produced within the field desorption-charge reduction region. Control of the abundance of the gas phase reagent ions is desirable to allow for selectable adjustment of the charge-state distribution of the analyte ions comprising the output of the ion source of the present invention. In a more preferred embodiment the corona discharge comprises an electrically biased wire electrode positioned close enough to a metal disc held at ground or substantially close to ground. The wire electrode and the metal disc are arranged in a point to plane geometry and separated by a distance sufficiently close to create a self-sustained electrical gas discharge. In another exemplary embodiment, the reagent ion source comprises a plurality of corona discharges. Other reagent ion sources useful in the present invention include but are not limited to an arc discharge, a plasma, a thermionic electron gun, a microwave discharge, an inductively coupled plasma and a laser or other source of electromagnetic radiation. In another exemplary embodiment, the reagent ion source comprises an externally housed flowing reagent ion source cooperatively coupled to the field desorption-charge reduction region and capable of providing a flow of reagent ions into the field desorption-charge reduction region.
In the present invention, the reagent ion source is substantially surrounded by a shield element for substantially confining the electric field, electromagnetic field or both generated by the reagent ion source. Accordingly, the shield element defines a shielded region wherein fields are minimized and in which charge reduction occurs. In an exemplary embodiment, the field desorption-charge reduction region is within the shielded region. In a preferred embodiment, a wire mesh screen held at an electric potential close to ground is positioned in a manner to substantially surround the reagent ion source and functions to substantially confine electric fields and/or electromagnetic fields generated. In another preferred embodiment, the shield is grounded. As a consequence of the presence of a shield, only one polarity of ion generated by the corona discharge is able to pass into the shielded region and interact with charged droplets and/or analyte ions. It is believed that this is due to the effect of electric fields generated by application of either positive or negative voltages to the first element of the corona discharge. Application of a negative voltage to the first biased corona discharge element results in the passage of negatively charged reagent ions into the shielded region and application of a positive voltage to the first biased corona discharge element results in passage of positively charged reagent ions into the shielded region.
The distance between the charged droplet source and the reagent ion source is selectively adjustable in the ion source of the present invention. In a preferred embodiment, the charged droplet source and/or the reagent ion source is moveable along a central chamber axis to permit adjustment of this dimension. It is believed that variation of this distance affects the field desorption conditions and extent of field desorption achieved. Accordingly, changing the distance between droplet source and reagent ion source is expected to affect the total output of the ion source of the present invention. Larger distances between droplet source and reagent ion source tend to allow for a greater extent of field desorption than shorter distances and, hence, tend to result in greater net ion production. In addition, variation of the distance between droplet source and reagent ion source may also affect field desorption conditions by changing the distribution of charge at the surface of the charged droplets. A smaller distance between droplet source and reagent ion source may lead to greater reagent ion/charged droplet interaction, thereby attenuating the charge on the droplet""s surface by charge scavenging. Scavenging of charge on the surface of the droplets is believed to have several effects on the field desorption process. First, charge scavenging can cause a net reduction in the extent and/or rate of field desorption of ions. Second, it may result in generation of analyte ions with a lower charge state distribution than that observed in the absence of charge scavenging.
The present invention may be utilized to generate a continuous or pulsed stream of analyte ions comprising negative ions, positive ions or both. In a preferred embodiment, the ion source of the present invention generates an output of gas phase analyte ions comprising substantially of singly charged ions and/or doubly charged ions. More preferably for certain applications, an ion source of this invention generates an output consisting essentially of singly and/or doubly charged ions. In particular, the present invention is highly suitable for generating singly charged ions and/or doubly charged ions from high molecular weight compounds in liquid samples. For example, the present invention may be used to produce singly and/or doubly charged gas phase ions from liquid samples containing at least one oligonucleotide and/or oligopeptide.
Alternatively, for certain applications an ion source of the present invention is useful for producing an output comprising multiply charge ions of a selected charge distribution. For example, singly charged analyte ions generated from chemical species with very high molecular weights can possess mass to charge ratios outside the detectable range of conventional mass spectrometers. Accordingly, the capability of the present invention to generate analyte ions of a selected multiply charged state from such chemical species permits the ion source of the present invention to generate detectable ions from chemical species with masses that extend beyond the mass range of conventional mass spectrometers.
Although the ion source of the present invention may be used to generate ions from any chemical species, it is particularly useful for generating ions from high molecular weight compounds, such as peptides, oligonucleotides, carbohydrates, polysaccharides, glycoproteins, lipids and other polymers. In addition, the ion source of the present invention may be utilized to generate gas phase analyte ions which possess molecular masses substantially similar to the molecular masses of the parent chemical species from which they are derived while present in the liquid phase. Most preferably for certain applications, the present invention may be utilized to generate singly and or doubly charged gas phase analyte ions possessing substantially similar molecular masses to the chemical species from which they are derived while present in the liquid phase. Accordingly, the present invention comprises an ion source causing minimal fragmentation to occur during the ionization process. In addition, the present invention provides methods of reducing the fragmentation of gas phase ions generated by electrospray ionization.
Alternatively, the ion source of the present invention may be used to induce and control analyte ion fragmentation by selectively varying the extent of multiple charging of the gas phase analyte ions generated. Gas phase ion fragmentation is typically a consequence of the substantially large electric fields generated upon formation of highly multiply charged gas phase ions. The occurrence of fragmentation may be useful in determining the identity and structure of chemical species present in liquid samples, the condensed phase and/or the gas phase. Accordingly, the ion source of the present invention maybe used to induce fragmentation of gas phase analyte ions by operating under experimental conditions that yield an output comprising multiply charged gas phase analyte ions in a selected charged state. In addition, an ion source of the present invention is capable of controllably adjusting the charge-state distribution of gas phase analyte ions to provide reproducible control over the gas phase ion fragmentation conditions. The ability to control fragmentation conditions is beneficial for the determination of analyte identity, structure and composition. Accordingly, the present invention provides a method of probing analyte identity and structure via controllable fragmentation.
In a preferred embodiment, the charge-state distribution of the gas phase analyte ions generated by the devices and methods of the present invention is adjustable by: 1.) varying the concentration of electrons and/or reagent ions generated within the field desorption region and 2.) by controlling the residence time of charged droplets and/or analyte ions in the field desorption-charge reduction region. The concentration of electrons and/or reagent ions generated in the field desorption region may be varied, for example, by adjusting the rate of electron and/or reagent ion production by the reagent ion source. Higher concentrations of reagent ions in the field desorption region results in an increase in the extent of charge reduction and lower concentrations of reagent ions results in a decrease in the extent of charge reduction. Control of the residence time of charged droplets and/or analyte ions in the field desorption-charge reduction region may be achieved, for example, by varying the linear flow rate of bath gas through the field desorption-charge reduction region, by adjusting the length of the field desorption-charge reduction region or both. In addition, it is believed that varying the charge-state distribution of the reagent ions generated within the field desorption region may also affect the charge-state distribution of analyte ions generated by the ion source of the present invention. It is believed that the charge-state distribution of the reagent ions in the field desorption-charge reduction region may be selectively adjusted by varying the operating conditions and type of reagent ion source employed. Accordingly, the present invention provides a means of producing ions from liquid samples in which the charge state distribution of the ions produced may be selectively controlled.
In a preferred embodiment, the ion source of the present invention comprises a source of ions whereby ionization processes and charge reduction processes are independently adjustable. Accordingly, the invention is not limited to any one means of ion formation and includes the combination of any ionization method capable of generating gas phase ions from liquid samples with the charge reduction methods described. This arrangement provides independent control of the charge-state distribution attainable without affecting the efficiency of the ion formation process employed. This characteristic of the present invention allows for efficient production of ions of varying charge-state distribution over a wide range of experimental conditions. Also this characteristic enables the methods of charge reduction of the present invention to be employed in combination with virtually any source of gas phase ions, charged droplets or both.
In another embodiment, the electrically charged droplet source is operationally coupled to an online purification system to achieve solution phase separation of solutes in a sample containing analytes prior to gas phase analyte ion formation. The online purification system may be any instrument or combination of instruments capable of online liquid phase separation. Prior to droplet formation and subsequent gas phase analyte ion production, sample containing solute is separated into fractions which contain a subset of species (including analytes) of the original solution. For example, separation may be performed so that each analyte is contained in a separate fraction. This configuration allows for ionization and charge reduction experimental conditions to be optimized for each separated fraction and/or individual analyte in the sample as it elutes from the liquid phase separation apparatus into the droplet source. The application of such separation techniques may significantly simplify sample analysis. In addition, the methods and devices of this preferred embodiment allow for formation of droplets that preferentially contain enhanced concentrations of analytes present in solution. Online purification methods useful in the present invention include but are not limited to high performance liquid chromatography, capillary electrophoresis, liquid phase chromatography, super critical fluid chromatography and/or microfiltration techniques. This preferred embodiment is particularly useful for purification and separation of samples containing one or more oligopeptide and/or oligonucleotide analytes prior to gas phase analyte ion production. Alternative embodiments include combinations of a plurality of online purification systems cooperatively coupled to the ion source of the present invention.
In another preferred embodiment, the ion source of the present invention is capable of simultaneously producing gas phase analyte ions of positive and negative polarities. These embodiments utilize reagent ion sources that generate both positive and negative gas phase reagent ions and allow both to interact with the stream of charged particles and/or gas phase analyte ions in the field desorption-charge regulation region. Positively and negatively charged reagent ions are formed in a periodic fashion and/or simultaneously in a manner which enables them to interact with charged particles and gas phase analyte ions in the field desorption-charge reduction region. This preferred embodiment allows for generation and charge-state reduction of analyte ions of either polarity. In addition, these embodiments may potentially serve as a means of re-ionizing analyte ions or droplets that undergo complete neutralization in the field desorption-charge reduction region. This may be accomplished by ion-molecule reactions between gas phase analyte ions and a bipolar reagent ion gas.
In an exemplary embodiment, the reagent ion source comprises a radio-frequency corona discharge comprising a first electrically biased element capable of oscillating between positive and negative voltages and a second element held at ground or near ground. The radio-frequency corona discharge provides a periodic source of positively and negatively charged reagent ions to the field desorption-charge reduction region. In another exemplary embodiment, the ion source of the present invention comprises a plurality of corona discharges. In this embodiment, the reagent ion source comprises at least one positive mode corona discharge, comprising a first electrically biased element held at a positive voltage and a second element held at ground or substantially close to ground, and at least one negative mode corona discharge, comprising a first electrically biased element held at a negative voltage and a second element held at ground or substantially close to ground. Negative and positive corona discharges are positioned downstream of the charged droplet source and individually surrounded by a shield element. The combination of positive and negative corona discharges provides simultaneous generation of positive and negative reagent ions in the field desorption-charge reduction region. It should be noted that any ion source capable of providing gas phase reagent ions of both positive and negative polarity to the field desorption-charge reduction region is useable in the present invention.
The present invention also comprises methods and devices for generating ions from gas phase neutral compounds generated from liquid samples. In an exemplary embodiment, electrically charged and/or neutral droplets are generated, entrained into a flow of bath gas and passed through an ionization region wherein neutral species are released into the gas phase. Within the ionization region gas phase neutral analytes undergo ion-neutral chemical reactions ionizing the gas phase neutral analytes thereby generating a flow of gas phase analyte ions. In this manner, gas phase neutral analytes are converted into gas phase analyte ions with an adjustable charge-state distribution. In a preferred embodiment, the output of the ion source of the present invention comprises singly charged ions, doubly charged ions, or both, generated from gas phase neutrals. Similarly, the present invention also comprises methods and devices for generating charged droplets from a stream of neutral droplets. In this embodiment, neutral droplets interact with reagent ions generated by the reagent ion source. Ion-droplet reactions result in charge accumulation on the droplets resulting in an output comprising a stream of charged droplets with a selectively adjustable charge state distribution.
In another embodiment, the ion source of the present invention is operationally coupled to a device capable of classifying and detecting charged particles. This embodiment provides a method of determining the composition and identity of substances which may be present in a mixture. In an exemplary embodiment, the ion source of the present invention is coupled to a mass analyzer and provides a method of identifying the presence of and quantifying the abundance of analytes in liquid samples. In this embodiment, the output of the ion source is drawn into a mass analyzer to determine the mass to charge ratios (m/z) of the ions generated from dispersion of the liquid sample into droplets followed by subsequent charge reduction. In an exemplary embodiment, the ion source of the present invention is coupled to a time of flight mass spectrometer to provide accurate measurement of m/z for compounds with molecular masses ranging from about 1 to about 30,000 amu. Other exemplary embodiments include, but are not limited to, ion sources operationally coupled to quadrupole mass spectrometers, tandem mass spectrometers, ion traps or combinations of these mass analyzers. Charge reduction conditions may be systematically varied during sampling to achieve optimal mass analysis for each analyte in a complex mixture because the present invention comprises a tunable ion source capable of varying charge reduction conditions as a function of time.
Alternatively, the ion source of the present invention may be coupled to a device capable of classifying and detecting ions on the basis of electrophoretic mobility. In an exemplary embodiment, the ion source of the present invention is coupled to a differential mobility analyzer (DMA) to provide a determination of the electrophoretic mobility of ions generated from liquid samples. This embodiment is beneficial because it allows ions of the same mass to be distinguished on the basis of their electrophoretic mobility.
The ability to generate a stream of gas phase analyte ions substantially comprising singly and/or doubly charged ions significantly enhances the utility of the present invention for the identification and quantification of analytes in liquid samples. The mass spectra obtained in electrospray discharge in the absence of charge reduction typically comprise a plurality of peaks attributable to each analyte detected. In contrast, mass spectra attained for samples containing complex mixtures of oligonucleotides and/or oligopeptides employing the present invention may be greatly simplified by charge reduction to substantially comprise single or double peaks attributable to each analyte present in a liquid sample. Accordingly, charge reduced mass spectra tend to be much easier to assign and quantify by persons of ordinary skill in the art of mass spectrometry. In addition, the reduced fragmentation characteristic of the ion source of the present invention also enhances the application of the ion source for analyte identification and quantification by decreasing chemical noise and increasing the intensities of mass spectral peaks easily assignable to parent analyte species.
The present invention also comprises methods for preparing gas phase analyte ions from a liquid sample, containing chemical species in a solvent, carrier liquid or both, wherein the charge-state distribution of the gas phase analyte ions prepared may be selectively adjusted. In a preferred embodiment, the method of preparing gas phase analyte ions comprising the steps of: (1) producing a plurality of electrically charged droplets of the liquid sample in a flow of bath gas; (2) passing the flow of bath gas and droplets through a field desorption-charge reduction region of selected length, wherein at least partial evaporation of solvent, carrier liquid or both from droplets generates gas phase analyte ions and wherein the charged droplets, analyte ions or both remain in the field desorption-charge reduction region for a selected residence time; (3) exposing the droplets, gas phase analyte ions or both to electrons, reagent ions or both generated from bath gas molecules by a reagent ion source that generates an electric field, electromagnetic field or both and is surrounded by a shield element that substantially confines the electric field, electromagnetic field or both generated by the reagent ion source defining a shielded region wherein fields generated by the reagent ion source are minimized, wherein the electrons, gas phase reagent ions or both react with said droplets, charged droplets or both within at least a portion of the field desorption region to reduce the charge-state distribution of the analyte ions in the flow of bath gas thereby generating gas phase analyte ions having a selected charge-state distribution; and (4) controlling the charge-state distribution of said gas phase analyte ions by adjusting the residence time of droplets, analyte ions or both, the abundance of electrons, reagent ions, or both, the type of bath gas, the type of reagent ion or both or any combinations thereof. Optionally, to comprise a method for determining the identity and concentration of chemical species in a liquid samples, the following step may be added to those provided above; (5) analyzing said gas phase analyte ions with a charged particle analyzer.
In addition, the present invention also comprises methods of reducing the fragmentation of gas phase ions generated from electrospray discharge of liquid samples. Smith et al., Mass Spectrometry Reviews, 10, 359-451 (1991) describe the fundamental principles and methods of electrospray ionization and is incorporated in this application in its entirety by reference. A preferred method of reducing fragmentation of the present invention comprises the steps of: (1) producing a plurality of electrically charged droplets from a liquid sample in a flow of bath gas by electrospray discharge; (2) passing the flow bath gas containing the droplets through a field desorption-charge reduction region of selected length, wherein at least partial evaporation of solvent, carrier liquid or both, from droplets generates gas phase analyte ions and wherein the charged droplets, analyte ions or both remain in the field desorption-charge reduction region for selected residence time; (3) exposing the droplets, gas phase analyte ions or both to electrons, reagent ions or both generated from bath gas molecules by a reagent ion source that generates an electric field, electromagnetic field or both and is surround by a shield element that substantially confines the electric field, electromagnetic field or both generated by the reagent ion source defining a shielded region wherein fields generated by the reagent ion source are minimized, wherein the electrons, gas phase reagent ions, or both, react with the droplets, charged droplets or both, within at least a portion of the field desorption region to reduce the charge-state distribution of the analyte ions in the flow of bath gas thereby generating gas phase analyte ions having a selected charge-state distribution; and (4) controlling the charge-state distribution of said gas phase analyte ions by adjusting the residence time of droplets, analyte ions or both, the abundance of electrons, reagent ions, or both, the type of bath gas, the type of reagent ion or both or any combinations thereof.
The invention is further illustrated by the following description, examples and drawings.